Cells interact with biochemically and structurally distinct forms of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat (2D) cell culture substrates? 2. Are there mechanisms for regulatory cross-talk between different types of extracellular matrix? We have been exploring which elements of the classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. We have identified a variety of differences in cell migration and signaling between 2D and 3D environments, but also in cellular responses to different 3D matrix environments. Reliable, reproducible characterization of cell interactions with adjacent collagen fibrils requires careful attention to methods of collagen labeling by fluorophores, polymerization into a fibrillar meshwork, and analysis by confocal microscopy, for which detailed methods were recently published. Human fibroblast migration in 3D collagen microenvironments requires myosin II, whereas this non-muscle myosin is not required when the same cells migrate on barrier-free 2D surfaces. Myosin II contractility appears to contribute to a novel mode of cell migration characteristic of locomotion in a 3D environment, which differs from the classical multi-step migration cycle described for 2D cell migration. The timing of protrusion of the leading edge relative to other intracellular movements differs, and both the nucleus and cell posterior tend to move relatively slowly and passively, which contrasts with the contraction phase of cell migration in 2D. For these analyses, we are tracking local movements of specific cellular features of migrating cells that include cell-matrix adhesions, the leading edge, nucleus, and trailing edge of cells compared to deformation of the local adjacent collagenous matrix. In studies nearing completion, we have discovered a new extracellular matrix regulatory cross-talk system in which basement membrane or its major molecular components induce rapid, strikingly robust fibronectin accumulation and assembly into fibrils. Although it had been known for decades that fibronectin strongly accumulates at basement membranes in vivo, the mechanisms were not clear. This novel matrix assembly mechanism appears to involve active sliding of focal adhesions containing the alpha5-beta1 fibronectin receptor along the basement membrane, collagen IV, or laminin to generate fibronectin fibrils distal to the adhesion. This phenomenon occurs with multiple cell types, both normal non-malignant and cancer cells. The mechanisms are being identified -- they appear to involve cellular myosin II-dependent shortening of actomyosin-based stress fibers. The focal adhesions are drawn toward each other in a winch-like manner, generating robust fibronectin fibrils in their wake as they translocate.